Straight-through process development of up and downstream integration of monoclonal antibodies production using flocculation, AEX and one pass TFF

The monoclonal antibody (mAb) market has been presenting a significant growth rate in the last two decades, which increased the interest of biopharmaceutical companies in this product class. Many improvements have been achieved in the upstream processing of mAbs, leading to significant increases in bioreactor titers. However, the production costs are still high, especially due to downstream processing costs, which can represent a major part of the of overall production costs. Traditional mAb platform processes include a very selective, but high-cost Protein A (PrA) affinity chromatography as the first purification step (capture). Several approaches have been recently explored in order to replace PrA chromatography. In this work, we propose a new, low-cost strategy for integrating clarification and capture step for mAbs using flocculation followed by a straight-through process with single-pass tangential flow filtration (TFF) and suspension anion-exchange (AEX) chromatography. First, the recombinant anti-IL8 mAb were produced by CHO-DP12 cells (ATCC, USA) in shake flasks at 180 rpm and 37°C using TC-LECC medium (Xell, Germany). After harvest, cells were flocculated using 5 pg per total cells at pH 6.5, allowing 15 min for settling of cells. Subsequently, the resulting supernatant and a Q-Sepharose resin (GE, Sweden) were pumped in equal amounts to a vessel, where a residence time for AEX adsorption of 15 min was applied, with the aim of allowing contaminants to adsorb to the resin. The resulting supernatant/AEX resin suspension was pumped out of the vessel into a 0.22-µm hollow fiber system (GE, USA). The mAb was recovered in the permeate, whereas the AEX resin remained in the retentate and could undergo elution, regeneration and sanitization for reusing. Two process variations were evaluated (Table 1), which were combined resulted in 6 different process strategies: (i) the ratio of clarified supernatant to AEX resin; (ii) the use of a device for cell/flocs retention named inclined lamella settler (Biotechnology Solutions, USA) and depth filter Clarisolve (Merck, USA), both were placed after the flocculation step to ensure a cleaner supernatant and to allow reducing the cell settling time. The integrated clarification-capture process showed to be simple and fast. Steady-state conditions were obtained during adsorption and filtration for all conditions studied. The average recovery of mAb during the steady-state was 48.5% ± 2%, which means a loss of approximately 3% of mAb product, since it was 2-fold diluted by the 1:1 mix with the resin suspension. However, considering the overall process, from start to final permeate recovery, global yields between 61% and 90% were obtained. These results are mainly related to the void volume of inclined lamella settler. The best global recovery (90.4%) was obtained when the depth filter was included in the process. Regarding impurities removal, in all 6 process strategies evaluated more than 85% of DNA was removed, and approximately 70% of HCP removal could be achieved when depth filter was used. Taking into account that two different supernatant/AEX resin ratios tested, a lower supernatant/resin ratio (41) provided a higher DNA clearance (86 fold), compared to less than one third of this clearance when sample/resin ratio was doubled to 82. Please click Additional Files below to see the full abstract

Clarification and capture of a CHO-derived monoclonal antibody through flocculation and AEX processes

Flocculants have been widely used in the food and chemical industry, as well as in wastewater clarification. More recently, studies have shown the applicability of these agents in the clarification of mammalian cell suspensions as an alternative e.g. to centrifugation and microfiltration1,2. Flocculants present several advantages such as low cost, process simplicity and facility to run under continuous mode. On the other hand, some flocculants have shown toxicity and thus their total clearance has to be demonstrated. In this work, the cationic flocculants chitosan and poly diallyldimethylammonium chloride (PDADMAC) have been investigated and compared for the clarification of suspensions of CHO cells producing a recombinant humanized monoclonal antibody. Anion exchange (AEX) adsorption has been then evaluated for its capacity to remove host cell protein (HCP) and host cell DNA (HCDNA), with the final aim of in the future developing an integrated train of clarification and AEX capture within a tangential flow filtration (TFF) device. CHO-DP12 cells (ATCC) were cultivated in batch mode in shake flasks at 37 ºC and 185 rpm using the animal component free medium TC-LECC (Xell AG, Germany), reaching approximately 10 million cells/mL and 0.2 g/L mAb. After cultivation, 20 mL of cell suspension were put in contact with flocculants under different conditions of pH and of flocculant concentration, for 30 min at 100 rpm at room temperature. The samples were then let to sediment for 90 min and the turbidity was measured for 90 min at 600 nm. The AEX resin Q-Sepharose (GE Healthcare, Sweden) was equilibrated at pH 6.5 prior to adsorption studies with the clarified supernatant from the flocculation step. Adsorption was carried out in 1.5-mL tubes by mixing the resin at a 1:10 volumetric ratio with different dilutions of the clarified supernatant for up to 120 min at room temperature under agitation at 1200 rpm. Samples were monitored for IgG (PrA-HPLC), total proteins (BCA) and DNA (picogreen). Regarding flocculation, when chitosan was used, no flocculation was observed in the concentration range of 0.01%-0.2% (10 to 200 pg per total cells), but did occur at lower flocculant concentrations. The best condition for chitosan was thus obtained at a concentration of 0.005% (5 pg per cell) and pH 6.5, resulting in 93.5% ± 1.1% IgG recovery and 98.5% ± 1.3% host cell DNA (HCDNA) removal. PDADMAC provided recoveries higher than 90% at concentrations between 0.0225% and 0.090%, with the latter concentration yielding an IgG recovery of 95.5% ± 2.4% and a HCDNA removal of 98.5%. For both agents, 30 min were sufficient to sediment all the flocculated particles under these best conditions. Under the selected conditions for chitosan flocculation (pH 6.5 and 5 pg per cell), AEX studies were performed in order to determine the adsorption capacity at equilibrium and the residence time needed to maximize removal of HCP and DNA from the clarified supernatant. Equilibrium was reached after 10 min, and adsorption capacity at equilibrium was 11 mg/mL for total proteins of and 15 mg/mL for DNA. Although at pH 6.5 IgG adsorption was not expected to occur, approximately 5 mg/mL were adsorbed (as measured by Protein A-HPLC). Further studies are ongoing to improve IgG recovery in the adsorption step, but these first results show that these simple, low-cost techniques can be used to obtain a cleaner material for further purification steps. The next steps of this work will focus on integrating flocculation, AEX and TFF to obtain a particulate free stream. 1. Riske, F. et al. The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery. 128, 813–823 (2007). 2. McNerney, T. et al. PDADMAC flocculation of Chinese hamster ovary cells : Enabling a centrifuge-less harvest process for monoclonal antibodies. 862, 413-427 (2015)

Integrated & continuous processing: A proven solution to tackle gene therapy manufacturing challenges

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Laboratory scale continuous linear purification as a development tool for recombinant blood protein processing, using chromatographic resins and membranes

Continuous processing offers significant advantages for the processing of unstable recombinant products such as therapeutic plasma proteins. As such we have established a development platform to assess potential purification steps as part of a continuous linear process using a standard AKTA Explorer. Following a consistent format of IEX membrane to HIC membrane to affinity resin, we were able to rapidly investigate multiple purification pathways for a recombinant blood protein. Using membranes as the first two steps enables a 3-step purification process to be carried out in 3 to 4 hours, with a total turnaround of 5 to 6 hours including regeneration and re-equilibration, at laboratory scale and depending on the specific pathway. This development method allowed for a direct comparison between multiple purification pathways, assessing overall recovery, pathway consistency, product quality and product purity over the 3 steps. Ligands tested include cation & anion exchangers, phenyl, phenyl boronate and an immobilised affinity peptide. It is envisaged that once a purification pathway has been decided upon, processing speed could be around 1 L per day without significant scale-up

Production and purification of a recombinant fragment of pneumococcal surface protein A clade 3 (PspA3) from Streptococcus pneumoniae in Escherichia coli.

A proteína A de superfície de pneumococo (PspA) é indispensável para a virulência da bactéria e foi escolhida para a elaboração de uma nova vacina conjugada contra S. pneumoniae. Para tanto foi desenvolvido um processo industrial de produção e purificação do fragmento recombinante da PspA clado 3 em E. coli. Cultivos descontínuos alimentados foram estabelecidos com glicose ou glicerol em reator de 5L, obtendo-se 62g/L de células secas e 3g/L de PspA3. As células foram lisadas por homogeneizador contínuo de alta pressão com eficiência de 96,7%. A centrifugação foi definida como etapa de clarificação. A sequência cromatográfica troca aniônica seguida de afinidade por Ni+2 rendeu os melhores resultados de pureza (81%) e recuperação (70%). A cromatografia de troca catiônica foi selecionada como terceira etapa do processo, definindo assim um processo de produção e purificação escalonável que possibilitou a obtenção de PspA3 com alto grau de pureza (90%).The pneumococcal surface protein A (PspA) is indispensable for virulence of S. pneumoniae and it was the first choice as carrier for a new conjugated vaccine against S.pneumoniae. Hence, the purpose of this work was to develop an industrial production and purification process of a recombinant fragment PspA clade 3 (rfPspA3) in E. coli. Fed-batch cultivations in 5 L bioreactors with defined medium were carried out using glucose or glycerol as carbon sources. It was obtained 62 g/L of dry cell weight and 3 g/L of rfPspA3. Cells were disrupted with 96.7% of efficiency by high pressure continuous homogenizer. Centrifugation was defined for the clarification step. The sequence with Q- followed by IMAC-Sepharose yielded the best purity and recovery of rfPspA3 (81 and 70%, respectively). Cation exchange was chosen for the last chromatography. In conclusion, an industrial production and purification process was developed and rfPspA3 was obtained with high purity (90%)

Humoral immune response of a pneumococcal conjugate vaccine : capsular polysaccharide serotype 14-Lysine modified PspA

Polysaccharide-protein conjugates are so far the current antigens used for pneumococcal vaccines for children under 2 years of age. In this study, pneumococcal surface protein A (PspA) was used as a carrier protein for pneumococcal capsular polysaccharide serotype 14 as an alternative to broaden the vaccine coverage. PspA was modified by reductive amination with formaldehyde in order to improve the specificity of the reaction between protein and polysaccharide, inhibiting polymerization and the gel formation reaction. In the synthesis process, the currently used activator, 1-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloride (EDAC) was substituted for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). BALB/c mice were immunized with either the PS14-mPspA conjugate or the co-administered components in a three dose regimen and sera from the immunized animals were assayed for immunity induced against both antigens: PS14 and mPspA. Modification of more than 70% of lysine residues from PspA (mPspA) did not interfere in the immune response as evaluated by the anti-PspA titer and C3 complement deposition assay. Sera of mice immunized with conjugated PS14-mPspA showed similar IgG titers, avidity and isotype profile as compared to controls immunized with PspA or mPspA alone. The complement deposition was higher in the sera of mice immunized with the conjugate vaccine and the opsonophagocytic activity was similar for both sera. Conjugation improved the immune response against PS14. The anti PS14 IgG titer was higher in sera of mice immunized with the conjugate than with co-administered antigens and presented an increased avidity index, induction of a predominant IgG1 isotype and increased complement deposition on a bacteria with a surface serotype 14. These results strongly support the use of PspA as carrier in a conjugate vaccine where both components act as antigens